In light of global environmental protection, the recent most important issue is to construct circulating social systems that can be maintained and continued. In such a social situation, much effort has also been made to develop recycling techniques for plastic wastes. Recycling techniques for plastic wastes are divided into two major types: physical approach (thermal recycling, material recycling) and chemical approach (chemical recycling). Among them, the former physical treatment has already been made practical for PET or other resins on a commercial basis because it is relatively simple and cost-effective. However, this approach cannot avoid quality loss due to repeated use and hence the resulting recycled products will have limited applications.
In the case of chemical recycling, on the other hand, waste plastics are chemically degraded into monomers or oligomers, which are then collected and used as source materials for resynthesis of new plastics. This approach allows production of plastic products completely comparable to primary products, and causes no quality loss. In light of these facts, products with chemical recycling in mind have recently been developed and a part of them has already been on the market.
Moreover, chemical recycling is also effective as a treatment technique for biodegradable plastics which have been spreading rapidly in recent years. In the future, biodegradable plastics will constitute nearly half of the total yield of plastics, and it is also expected that attention will be given to the development of efficient techniques for their recycling in the future. Biodegradable plastics currently in circulation are almost exclusively polyester-based plastics. This means that monomer recycling can be very easily achieved for these plastics because their monomer components such as organic acids and polyhydric alcohols are joined via ester bonds sensitive to hydrolysis.
However, a large problem arises when actually attempting recycling because wastes comprise multiple types of materials in admixture. Although there is a cry for separated collection of wastes, it will actually be impossible to completely achieve separated collection when taking into account the awareness of people who discard wastes as well as the time and effort required for separated collection. In particular, plastic products generally use a plurality of different plastics in combination, and currently used techniques do not enable the separation of all plastic wastes according to plastic types. For this reason, under present circumstances, recycling is limited to wastes which are easy to separate and collect, regardless of recycling techniques.
Likewise, the chemical recycling of biodegradable plastics is also very disadvantageous in terms of costs because standard chemical degradation such as acid or alkali degradation produces monomers in a mixture form, which require many processes for their purification.
To overcome this problem, a new process has been proposed in which enzymes are used for chemical recycling of plastics. As to merits resulting from the use of enzymes, the substrate specificity of enzymes may be the most excellent feature although it is also important in that reactions can be carried out at normal temperature and under normal pressure, thereby saving energy costs and requiring no organic solvent responsible for environmental pollution. In general, enzymes have substrate specificity and clearly select their target substrates. Thus, a combination of enzymes, each being reactive to only a certain specific plastic, allows efficient extraction of high purity monomers from plastic wastes in a mixture form, without requiring any separation process. In general, bioprocesses require high costs and hence are disadvantageous in this point without any doubt, but it is a great merit to achieve extraction of high purity monomers without separation. Particularly, also in view of the fact that biodegradable plastics are degraded by the action of enzymes secreted by microorganisms in the natural world, it can be expected to develop a process using enzymes derived from such degrading bacteria.
To establish enzymatic recycling, the premise is the presence of a strong plastic-degrading enzyme having high substrate specificity. In particular, since plastic wastes are practically discarded in solid form such as chips and/or blocks, bacteria which degrade solids are particularly important.
Enzymes previously known for their ability to degrade polyester-based solid plastics include enzymes that degrade polyhydroxyalkanoate (PHA), i.e., PHA depolymerases. PHA is a natural polyester produced by microorganisms and has been used as a biodegradable plastic from a long time ago. Since PHA is an energy storage substance inherent to bacteria, there is of course a metabolic system where PHA is degraded to produce energy. Thus, many bacteria including Pseudomonas spp. are known to have the ability to degrade PHA. On the other hand, however, such enzymes have little reactivity to any polyester-based plastic other than PHA.
Examples known as enzymes derived from unnatural plastic-degrading bacteria are those capable of degrading ester-based polyurethanes. Such enzymes are derived from Comamonas acidovorans and cleave ester bonds in ester-based solid polyurethanes to produce water-soluble monomers [Akutsu, Y., Nakajima-Kambe, T., Nomura, N., and Nakahara, T.: Purification and properties of a polyester polyurethane-degrading enzyme from Comamonas acidovorans TB-35. Appl. Environ. Microbiol., 64, 62-67 (1998); and JP 09-224664 A entitled “Method for polyurethane esterase purification and method for ester-based polyurethane degradation” (Applicant: Suzuki Motor Corporation; Inventors: Toshiaki Nakajima, et al.)].
Other polyester-based plastics include polylactic acid, polybutylenesuccinate (PBS), polybutylenesuccinate-co-adipate (PBSA) and polycaprolactone (PCL), each of which is a biodegradable plastic. Although there have been many reports of degrading bacteria for these biodegradable plastics, most of these reports were directed to degradation of emulsified or powdered plastics or thin films of micron order (Kim, D. Y., and Rhee, Y. H.: Biodegradation of microbial and synthetic polyesters by fungi. Appl. Microbiol. Biotechnol., 61, 300-308 (2003)). Uchida et al. have isolated Acidovolax delafieldii strain BS-3 which assimilates PBSA pellets as a sole carbon source JP 11-225755 A entitled “Biodegradable polymer-degrading enzyme and method for its preparation” (Applicant: Mitsubishi Chemical Corporation; Inventors: Toshiaki Nakajima, et al.); and Uchida, H., Nakajima-Kambe, T., Shigeno-Akutsu, Y., Nomura, N., Tokiwa, Y., and Nakahara, T.: Properties of a bacterium which degrades solid poly(tetramethylene succinate)-co-adipate, a biodegradable plastic. FEMS Microbiology Letters, 189, 25-29, (2000)], but there is no other report about bacteria capable of degrading solid pellets.
As for polylactic acid, there is a report of a degrading enzyme derived from actinomycetes Amycolatopsis sp. strain K104-1, but degradation in film form requires 48 hours or more and degradation of PBSA is not discussed in this report (Nakamura K, Tomita T, Abe N, and Kamio Y.: Purification and characterization of an extracellular poly(L-lactic acid) depolymerase from a soil isolate, Amycolatopsis sp. strain K104-1. Appl. Environ. Microbiol. 67, 345-353 (2001)). Likewise, a polylactic acid-degrading enzyme derived from Peanibacillus amylolyticus strain TB-13 [Akutsu-Shigeno, Y., Teeraphatpornchai, T., Teamtisong, T., Nomura, N., Uchiyama, H., Nakahara, T., and Nakajima-Kambe, T.: Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Peanibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli. Appl. Environ. Microbiol., 69, 2498-2504 (2003); and JP 2004-166540 A entitled “Novel plastic-degrading enzymes and genes encoding the enzymes” (Applicant: Japan Science and Technology Agency, Inventors: Toshiaki Nakajima, et al.)] also allows degradation of polybutylenesuccinate-co-adipate (PBSA), but the degradation is less successful and limited to the emulsion form.
On the other hand, as for polybutylenesuccinate-co-adipate (PBSA), there are many reports of degrading bacteria, and enzymes derived from mold strains have been partially purified. However, no attempt has been made to purify bacterial enzymes. A PBSA-degrading enzyme and cloning of its gene are reported for Acidovolax delafieldii strain BS-3 [JP 11-225755 A entitled “Biodegradable polymer-degrading enzyme and method for its preparation” (Applicant: Mitsubishi Chemical Corporation; Inventors: Toshiaki Nakajima, et al.); and Uchida, H., Y. Shigeno-Akutsu, N. Nomura, T. Nakahara, and Nakajima-Kambe, T.: Cloning and Sequence Analysis of Poly(tetramethylene succinate) Depolymerase from Acidovorax delafieldii Strain BS-3. J. Biosci. Bioeng., 93, 245-247 (2002)], but this enzyme has low degrading ability and allows only degradation in emulsion form, but not in film form.
In view of the foregoing, there are a limited number of reports on microorganisms capable of degrading plastics in film or pellet form, and further their enzymes are poorly known. To establish enzymatic recycling, there is a strong demand for enzymes capable of rapidly degrading solid plastics.
In addition to recycling, as proper treatment for biodegradable plastics, degrading bacteria have a potential for being added to a kitchen refuse treater and/or for composting. In this case, such bacteria are desired to successfully degrade plastics even in a nutritious environment. However, many previous degrading microorganisms use the plastics as a sole carbon source, and hence their degrading ability is significantly decreased or lost in the presence of other organic materials at high concentrations. There have been few cases to search for microorganisms allowing degradation in a nutritious environment.